Our objective is elucidation of mechanisms of xenobiotic metabolism in the regulation of the biological fate of foreign chemicals. The work has primarily been on azo dye carcinogens, some of which are frequently found in human environment. Rat liver microsomes metabolize butter yellow (dimethylaminoazobenzene, DAB) by oxidative and reductive pathways. The focus will be primarily on azoreductase inasmuch as this represents detoxication for DAB and other toxic azo dyes, and the mechanisms for azoreduction are not well understood. There are four aspects of the proposed research. (1) The form of cytochrome P-450 which catalyzes DAB azoreduction will be purified from rat liver microsomes. The technique includes affinity and ion exchange chromatography and more recent methods with preparative FPLC. Starting material will be microsomes from rats treated with clofibrate or similar hypolipidemic drug which selectively induce DAB azoreductase activity. (2) Studies on the purified enzyme will include kinetics, effects of inhibitors and structure-activity relationships. (3) The question of azoreduction sensitivity and insensitivity to oxygen will be addressed since reduction of certain azo dyes is inhibited by oxygen. Appearance of free radicals will be measured by EPR to test the theory that oxygen sensitivity is associated with a readily oxidized one electron reduced free radical intermediate during azoreduction. (4) The role of cytochrome b5 in DAB azoreduction will be investigated. Purified cytochrome will be prepared and used in purified reconstituted cytochrome P-450 systems. Antibodies will be prepared to cytochrome b5 and their effect on microsomal DAB azoreductase activity determined. (5) Studies will be performed in the perfused liver to determine the role of azoreduction in the hepatic fate of DAB. Biliary and urinary metabolites of DAB will be measured after treatment to induce or suppress DAB azoreduction.